References The following publications are cited in this application as superscript numbers : 1. Durance, T.D. 1994, "Isolation and thermal stability of lysozyme and avidin", In Egg Uses and Processing Technologies- New Development. editied by J. S. sim and S. Nakai, CAB International, Wallingford Oxon U. K.

2. Fasman G.D. 1989. Practical Handbook of Biochemistry and Molecular Biology CRC Press Inc. , Boca Raton, Florida p 555 3. Jolles and Jolles, 1984,"What's new in lysozyme research"Mol. and Cell. Biochem 63 : 165-189 4. Green, N. M. 1963."Avidin : The use of [14C] biotin for kinetic studies and for assay. Biochem J. 89:585-591 5. Green, N. M., 1975,"Avidin"Adv. Protein Chem 29 : 85-133 6. Korpela, et al., 1981."Biotin binding proteins in eggs of oviparous vertebrates" Experimentia 37:1065-1066

7. Villa A, 1996"Controlling malolactic fermentation (MLF) with lysozyme : application and results"Wine Spoilage Microbiology Conference March 1996,49-64 8. Korpela et al., 1984, "Avidin: a high affinity biotin-binding protein, as a tool and subject of biological research"Medical Biology 62 : 5-26 9. Amati et al.,"Lysozyme application to control malolactic fermentation : industrial trials" 46th Annual Meeting of the American Society for Enology and Viticulture, June 22-24 1995 Oregon Convention Center, Portland Oregon 10. Green et al., 1995,"Efficacy of lysozyme in preventing malolactic fermentation in Pino Noir wines" 46th Annual Meeting of the American society for Enology and Viticulture, June 22-24 1995 Oregon Convention Center, Portland Oregon 11. Shugar, D. 1952 Biochimica et Biophysica Acta 8:302-309 12. Sponholz, W. R. 1993"Wine Spoilage by Microorganisms"in Wine Microbiology & Biochemistry, ed G. H. Fleet, Harwood Academic Publishing, pages 396-400 13. Green, N. M., 1963 Biochem J. 89 : 599 13. Green, N. M., 1965 Biochem. J. 94:230 All of the above publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.

State of the Art There are three stages in the process of winemaking where microbiological contamination may occur and diminish the quality and acceptability of the final product. The first stage is the raw grapes, which can become infected with undesirable molds, yeasts and bacteria. The second stage occurs during fermentation. Yeasts naturally present on the fruit or originating in the winery

environment will contribute to the fermentation by Saccharomyces cerevisiae.

Growth of undesirable species at this stage may spoil the wine. The wine, after fermentation is not a stable product and if stored and handled inexpertly can become a growth substrate for a range of undesirable yeast and bacterial species.

In particular the yeast species Hanseniaspora uvarum (Kleockera apiculata), Metschnikowia pulcherrima, Hansenula anomala, Brettanomyces, Candida, Pichia and Zygosaccharomyces bailEi are known to cause spoilage of wine (Sponholz, W. R. 12) Accordingly, during vinification there is a need to prevent contamination of the must or wine by unwanted microorganisms. Current oenological technology uses an early drawing off of the wine, frequent decanting operations, clarification and filtration of the wine, storage at a low temperature and the use of appreciable quantities of sulphur dioxide to control growth of unwanted microorganisms.

Considerable doubts have arisen from the health standpoint with regard to the use of sulphur dioxide.

In addition, immediately after or during alcoholic fermentation there can be a secondary fermentation by the lactic acid bacteria Lactobacillus, Leuconostoc, and Pediococcus, the so-called malolactic fermentation. Malolactic fermentation causes decarboxylation of a malic acid molecule and the formation of a lactic acid molecule and a carbon dioxide molecule. This will cause a rise in the pH of the wine. The rise in pH may also result in bacterial and yeast proliferation with a consequent degradation of the wine's clarity, taste and bouquet.

Accordingly, it would be advantageous to identify antimicrobial agents useful in inhibiting the growth of unwanted microorganisms during the fermentation and storage of the wine.

In white wines and for some rose wines, the malolactic fermentation may cause an undesirable weakening of the bouquet and flattening of the taste. The use of lysozyme to inhibit the growth of bacteria responsible for malolactic

fermentation of wine has previously been suggested (Amati et al., 19959; Green et al., 1995'° ; Villa, A, 1996').

Lysozymes (muramidase : mucopeptide N-acetylmucamoylhydrolase ; 1, 4- (3- N acetylhexosaminodase, E.C. 3.2.1.17) are mucolytic enzymes which have been isolated from various sources and are well characterized. Egg white lysozyme is an enzyme that consists of 129 amino acids, cross-linked by 4 disulfide bridges (Jolles and Jolles 19843). Its molecular weight is approximately 14, 300 to 14, 600 daltons. The isoelectric point is pH 10. 5-10. 7. This polypeptide has muramidase and chitinase activity that degrades bacterial and yeast cell walls.

Avidin is a basic egg white tetrameric glycoprotein made up of four identical polypeptide subunits. The amino acid sequence of an avidin subunit contains approximately 128-129 amino acids, with alanine and glutamate at the amino and carboxyl ends, respectively, with a uncharacterized carbohydrate moiety. The molecular weight of the entire molecule is about 67, 000 daltons.

Each subunit has an intrachain disulphide bond (Korpela 1984). Avidin has a strong and specific affinity for the vitamin biotin and binds four molecules of biotin, one per subunit. The avidin-biotin interaction is characterized by a dissociation constant of approximately 10-l5 M, making it one of the strongest non- covalent ligand protein interactions known (Green, 19634). Lysozyme and avidin can be isolated together from the egg white (Durance, 1994').

SUMMARY OF THE INVENTION In view of the foregoing limitations and shortcomings of the prior art methods of inhibiting growth of microorganisms in wine, it is apparent that there still exists a need in the art for methods and compositions for inhibiting the growth of unwanted microorganisms in wine which methods do not require the application of potentially harmful chemicals.

This invention is directed to a process for inhibiting the growth of unwanted microorganisms in wine, which method comprises the addition of a composition comprising an effective amount of avidin to the wine under conditions whereby growth of microbes susceptible to avidin is inhibited. The composition may further comprise an effective amount of lysozyme for inhibition of the growth of microbes susceptible to lysozyme. Preferably, the microoraanism whose growth is inhibited is either Brettanomyces spp. or Kloeckera spp.

It has also been found the growth of the yeast genus Kloeckera spp. is inhibited by lysozyme. Accordingly, this invention is also directed to a process for inhibiting the growth of Kloeckera spp. in wine contaminated by Kloeckera spp. by the addition of an effective amount of lysozyme under conditions whereby growth of the Kloeckera spp. is inhibited.

This invention is also directed to a composition comprising from about 10 weight percent to about 90 weight percent avidin and from about 10 weight percent to about 90 weight percent lysozyme. The composition may additionally comprise from about 2 weight percent to about 25 weight percent of ovotransferrin.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the minimal inhibitory concentrations (MIC) of avidin for the three Brettanomyces strains.

FIG. 2 is a graph showing the minimal inhibitory concentrations (MIC) of Lysozyme type 7 for the three Brettanomyces strains.

FIG. 3 is a graph showing the minimal inhibitory concentration of lysozyme for the Kloeckera strain.

FIG. 4 is a graph showing the minimal inhibitory concentration of Lysozyme type 7 for the Kloeckera strain.

FIG. 5 is a graph showing the percentage of survival of Kloeckera at various concentrations of lysozyme.

FIG. 6 is a graph showing the percentage of survival of Kloeckera at various concentrations of Lysozyme type 7.

FIG. 7 is a graph showing the percentage survival of Brettanomyces cells at various concentrations of Lysozyme type 7.

DETAILED DESCRIPTION OF THE INVENTION When discussing such methods, the following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

"Avidin"is any enzyme capable of binding biotin with high affinity.

Preferably, avidin is the basic egg white tetrameric glycoprotein made up of four identical polypeptide subunits. The amino acid sequence of an avidin subunit contains approximately 128-129 amino acids. Active avidin has a specific affinity for and binds with four molecules of biotin, one per subunit.

It is contemplated that the avidin may be naturally occurring avidin purified from eggs, it may be obtained from prokaryotic or eucaryotic cells modified to produce avidin or it may be produced synthetically. Avidin is commercially available from Canadian Inovatech, Inc. , Abbotsford, B. C. Canada.

Without being limited to a theory, it is believed that avidin inhibits microbial growth by preventing uptake of biotin (Korpela et al., 19816, Green 19755).

Preferably, the activity of the avidin composition used in the antimicrobial compositions of this invention has an activity of about 0. 5 unit/mg to about 16 units/mg, and more preferably from about 5 units/mg to about 15 units/mg. One unit of avidin is defined as the amount of protein that will bind one microgram of d-biotin at pH 8. 9 (Green 1963l3).

"Lysozyme"used in the present invention is any lysozyme capable of degrading bacterial and yeast cell walls. Lysozymes (muramidase : mucopeptide

N-acetylmucamoylhydrolase ; 1, 4-P-N acetylhexosaminodase, E. C. 3. 2. 1. 17) are mucolytic enzymes which have been isolated from various sources and are well characterized. There are three classes of lysozymes, type c (chicken), type g (goose) and type v (viral).

Preferably, the lysozyme is type c egg white lysozyme. Egg white lysozyme is an enzyme that consists of approximately 129 amino acids, cross- linked by 4 disulfide bridges (Jolles and Jolles 19843). Its molecular weight is approximately 14, 300 to 14, 600 daltons. The isoelectric point is pH 10. 5-10. 7.

This polypeptide has muramidase and chitinase activity.

It is contemplated that the lysozyme may be naturally occurring lysozyme purified from eggs, it may be obtained from prokaryotic or eucaryotic cells modified to produce lysozyme or it may be produced synthetically. Lysozyme is commercially available from Canadian Inovatech, Inc. , Abbotsford, B. C. Canada.

Preferably, the lysozyme composition used in the anitmicrobial compositions of this invention has an activity of about 10, 000 units/mg to about 30, 000 units/mg, more preferably from about 15, 000 units/mg to about 24, 000 units/mg. There are a number of methods for determining the activity of lysozyme (Shugar, D. 1952 ") One method for determining the activity of a lysozyme solution is to determine the change in absorbance of a Micrococcus lysodeikticus culture after the addition of the lysozyme solution. As the lysozyme lyses the Micrococcus cell wall, the absorbance of the culture decreases with time. One unit of lysozyme is the amount of enzyme that causes a decrease in absorbance of 0. 001/min at 450 nm at pH 6. 2 at 25°C.

"Ovotransferrin"means any protein capable of acting like egg white ovotransferrin. Egg white ovotransferrin, also known as conalbumin, is a glucoprotein with a molecular weight of approximately 78,000 daltons. It has an isoelectric point of 6. 1. It contains two lobes connected by an a-helix. Each lobe is homologous and can bind an Fe3+ ion. The iron-binding site in each lobe is

situated between two sub-domains. The presence of bicarbonate ion may enhance the binding of iron to the molecule.

The term"effective amount"means that amount of an antimicrobial composition which is capable of inhibiting the growth of the microorganism. This amount will be determined by those skilled in the art based on the amount of wine to be treated, the time of treatment, the pH of the wine and the activity of the avidin or lysozyme to be used.

The terms"contact"or"contacting"mean the addition of the antimicrobial composition to the wine by standard methods in the wine making art.

The antimicrobial composition of the present invention may be a solid in the form of a cake, powder or granulates. Alternatively, the antimicrobial composition may be a liquid, gel or paste. The antimicrobial composition may be encapsulated in a micelle or liposome. It may be freeze dried or spray dried.

The antimicrobial compositions of the present invention may comprise an effective amount of avidin and/or an effective amount of lysozyme. They may also comprise an effective amount of ovotransferrin. Where the antimicrobial composition comprises an effective amount of avidin, preferably the concentration of avidin in the antimicrobial composition is from 5 % to about 100% by weight of the antimicrobial composition, more preferably the concentration is from about 10% to 90% by weight and most preferably the concentration is from about 20% to 80% by weight.

Where the antimicrobial composition of the present invention comprises an effective amount of lysozyme, preferably the concentration of lysozyme in the antimicrobial composition is from 5 % to about 100% by weight of the antimicrobial composition, more preferably the concentration is from about 10% to 80% by weight and most preferably the concentration is from about 20% to 80% by weight.

A preferred antimicrobial composition is a combination of an effective amount of avidin and an effective amount of lysozyme. Preferably, this composition comprises from about 5% to about 90% of avidin and from about 10% to about 95% lysozyme by weight. More preferably, this composition comprises from about 10% to about 80% avidin and from about 20% to about 90% lysozyme by weight.

The antimicrobial compositions of this invention may further comprise from about 2 % to about 25 % of ovotransferrin, more preferably from about 5 % to about 15% by weight.

One antimicrobial composition useful in this invention is Lysozyme-type 7, (commercially available from Canadian Inovatech, Inc. , Abbotsford B. C. Canada) which comprises approximately 10-25% lysozyme, 35-50% avidin, 20-35% ovalbumin, 5-20% ovotransferrin and other egg white proteins.

"Microorganisms susceptible to avidin"means any microorganism whose growth is inhibited by exposure to avidin. Such microorganisms may include fungi, bacteria and yeast. Examples of such microorganisms include, but are not limited to, the following : Brettanomyces spp., such as Brettanomyces anomales, Brettanomyces bruxellensis, Brettanomyces custerianus, Brettanomyces naardenensis, Botrytis spp., such as Botrytis cinerea, Candida spp., Dekkera spp., Hanseniaspora spp, Kloeckera spp. and Pickia spp. Preferably, the microorganism susceptible to avidin is Brettanomyces spp.

The"microorganisms susceptible to Iysozyme"means any microorganism whose growth in inhibited by exposure to lysozyme. Such microorganisms may include fungi, bacteria and yeast. Examples of such microorganisms include, but are not limited to, the following: Kloeckera spp., Botrytis cinerea, Lactobacillus spp. such as Lactobacillus hilgardii, Lactobacillus kunkeii, Lactobacillus brevis, <BR> <BR> <BR> Lactobacillus plantarum,, Leuconostoc oenos (Oenococcus oenos), Micrococcus lysodeikticus, Pediococcus spp. such as Pediococcus damnosus, Pediococcus dextrinicus, Pediococcus parvulus, Pediococcus pentocaceus, and Pediococcus

urinaeequi. More preferably, the organism susceptible to lysozyme is Kloeckera spp.

The"microorganisms susceptible to ovotransferrin"means any microorganism whose growth in inhibited by exposure to ovotransferrin. Such microorganisms may include fungi, bacteria and yeast.

One skilled in the art could determine which microorganisms are susceptible to a microbial composition comprising avidin, lysozyme or ovotransferrin by contacting the microorganisms with the antimicrobial composition and measuring the inhibition of growth of the microorganism.

The term"inhibition of growth"means that the number of viable cells of the microorganism is reduced by at least 30%, more preferably by at least 40% and most preferably by at least 50%. The percentage of inhibition of growth can be determined by a number of ways known in the art. Such methods include cell counting under a microscope using a hemacytometer after staining for viable cells and culture plate assays.

The methods of the present invention are applicable in general to any type of wine, such as, but not limited to red wine, rose and white wine. The term "must"means the mixture of grape juice, skins etc. resulting from pressing of the wine grapes. The term"wine"as used herein includes the term'must"and means the liquid which begins the process as"must"and which, as it proceeds through the filtration, fermentation and cellaring stages, becomes wine.

The compositions of this invention will typically contain additional components which are compatible with wine. Such components may include, for example, buffer materials for pH control and the like.

A buffer may be included in the antimicrobial composition in view of the fact that avidin and lysozyme are pH sensitive and thus, are desirably protected from potentially damaging variations in pH. It will be appreciated, however, that it is possible that the natural pH of the wine being treated, which is typically

acidic, will be within the acceptable pH range of the avidin or lysozyme being applied. In such instances a buffer may not be required.

When a buffer is employed, there may be used any buffer solution which is compatible with the avidin and/or lysozyme and which does not otherwise deleteriously affect the wine. Of course the buffer must also be a material which is known to be safely used with a foodstuff.

Water will often be used as a carrier for the avidin and lysozyme composition, but other conventional carriers may also be used.

Other additives may include preservatives such as potassium sorbate.

Methodology It should be understood that when the composition and method of this invention are employed, the composition is added to the wine and to any microbial contamination which may be present in the wine. In general, the compositions of this invention are applied to the wine in accordance with known techniques.

The anitmicrobial compositions of the invention can be added to the wine at any stage of the wine making process. The compositions may be added prior to fermentation to inhibit undesired microbial growth. The composition may also be added to the wine after alcoholic fermentation and before or during malolactic fermentation. The compositions may also be added during the aging or cellaring process. Preferably, the antimicrobial composition is added to the wine after fermentation and prior to cellaring.

In selecting a point in the wine-making process for treatment, other components in the wine may affect the amount of antimicrobial composition used.

The presence of bentonite in the wine may affect the activity of the avidin and lysozyme. Similarly the presence of SO2 may also affect the activity of the avidin and lysozyme. Accordingly, additional lysozyme and/or avidin may need to be added to the wine. A person skilled in the art could readily adjust the amount of antimicrobial composition added to the wine to ensure sufficient inhibition of growth of the microorganisms in the wine.

Upon addition of the compositions to the must or wine, the avidin and/or lysozyme acts to inhibit the growth of microorganisms susceptible to avidin and/or lysozyme. Similary, if the composition further comprises ovotransferrin, the ovotransferrin also acts to inhibit the growth of microorganisms susceptible to ovotransferrin.

The antimicrobial composition comprises lysozyme, the composition is added to the wine such that the final concentration of avidin in the wine is from about 0. lmg/L to about 1000 mg/L, more preferably from about 1 mg/L to about 500 mg/L. Where the antimicrobial composition comprises lysozyme, the composition is contacted with the wine such that the final concentration of lysozyme in the wine is from about 1 mg/L to about 5000 mg/L, more preferably from about 1 mg/L to about 2000 mg/L. Where the antimicrobial composition comprises ovotransferrin, the composition is contacted with the wine such that the final concentration of ovotransferrin in the wine is from about 0. 1 mg/L to about 1000 mg/L, more preferably from about 1 mg/L to about 500 mg/L.

Normally, the compositions are added to the wine at cellar temperature.

It is understood that the temperature of treatment should be compatible with wine production. Preferably, the temperature range for treatment will be between 5°C and 50°C and more preferably between 10°C and 40°C.

The avidin and lysozyme are effective within the pH range of wine.

Preferably the pH of the wine will be from about 2. 5 to about 7 and most preferably, the pH of the wine will be from about 2. 8 to about 5.

Utility The compositions and method of this invention are useful in the inhibition of growth of certain microorganisms during the fermentation and storage of wine.

The following examples are offered to illustrate this invention, and are not to be construed in any way as limiting the scope of this invention. Unless otherwise stated, all temperatures are in degrees Celsius.

EXAMPLES In the examples below, the following abbreviations have the following meanings. If an abbreviation is not defined it has its generally accepted meaning.

9 gram mg milligram g microgram L = liter ml = milliliter <BR> 1 = microliter <BR> au micron MIC = minimal inhibitory concentration ppm = parts per million rpm revolutions per minute Example 1 Inhibition of Microorganisms in Wine Each of the indicated microorganisms was grown in 5 ml of media at 22°C on a 180 rpm shaker. WL nutrient broth (DIFCO Laboratories, Inc. Detroit, MI), WLC froth (WL nutrient broth + 50 ppm cycloheximide) and Modified Rogosa (Izuagbe et al. , 1985, Applied Microbiology, 50:680-684) from fresh tomato juice were used.

A sequential series of concentrations of lysozyme, avidin or Lysozyme-type 7 (Canadian Inovatech, Inc. , Abbotsford BC Canada) were added to different microtiter wells containing 100 1 of media. The lysozyme powder used had an activity of 15000-24000u/mg. The avidin powder used had an activity of approximately 2 - 14 u/mg as determined by the Worthington method. A solution of approximately 104 cells/ml of each of the microorganisms was prepared by counting the cells from the 5 ml of growth media on a hemacytometer and diluting appropriately. 101 of the 104 cells/ml solution of test microorganisms was added

to each of the microtiter wells. The microtiter plates were incubated at 22 °C (room temperature) for 2 weeks, and observed on day 7 and day 14 for growth of the microorganism. 50, ul of the 104 diluted test strain was also plated on agar plates for a viable cell count.

The results are presented in Table 1 below.

Table 1 IM# Microorganism pH MIC + length of time Lysozyme Avidin Lysozyme type 7 106 Pediococcus ~5.4 31.3 ppm/ N/T N/T parvulus 2 weeks 107 Pediococcus ~5.4 7.8 ppm/ N/T N/T parvulus 2 weeks 147 Brettanomyces ~5.4 no inhibition/ 3.9 ppm/ 15.6 ppm/ strain 1 2 weeks 2 weeks 2 weeks 147 Brettanomyces ~3.4 no inhibition/ 125 ppm/ 250 ppm/ strain 1 2 weeks 2 weeks 2 weeks 148 Brettanomyces ~5.4 no inhibition/ 7.8 ppm/ 15.6 ppm/ strain 2 2 weeks 2 weeks 2 weeks 148 Brettanomyces 3. 4 no inhibition/125ppm/250 ppm/ strain 22 weeks 2 weeks 2 weeks 149 Brettanomyces ~5.4 no inhibition/ 7.8 ppm/ 15.6 ppm/ strain 3 2 weeks 2 weeks 2 weeks 149 Bretlanomycess ~3.4 no inhibition/125 ppm/62. 5 ppm/ strain 3 2 weeks 2 weeks 2 weeks 183 Leuconostoc ~5.4 2.0 ppm/ N/T N/T MCW 1 week

IM# Microorganism pH MIC + length of time Lysozyme Avidin Lysozyme type 7 184 Kloeckera ~5.4 62.5 ppm/ 250 ppm/ 62.5 ppm/ slows growth slows growth slows growth 1 week 1 week 2 weeks 125 ppm/500 ppm/ slows growth slows growth 2 weeks 2 weeks 184 Kloeckera ~3.4 500 ppm/250 ppm/62. 5 ppm/ slows growth slows growth slows growth 2 weeks 2 weeks 2 weeks 185 Lactobacillus ~5.4 no inhibition/ N/T N/T brevis 2 weeks 186 Pediococcus -5. 4 15. 6 ppm/N/T N/T sp. 2 weeks 189 Leuconostoc ~5.4 3.9 ppm/ N/T N/T oenos 1 week 196 Lactobacillus ~5.4 3.9 ppm/ N/T N/T kunkeei 1 week N/T = not tested Lysozyme is very effective in inhibiting Pediococcus spp. (50 ppm) (20 ppm), Leuconostoc oenos (10 ppm) and Lactobacillus kunkeei (5 ppm). The growth of Kloeckera was slowed down at 250-500 ppm of lysozyme.

Pure avidin and Lysozyme type 7 are very good at inhibiting the growth of Brettanomyces (5-250 ppm) and at slowing the growth of Kloeckera.

Example 2 Inhibition of Brettanomvees by avidin and Lysozyme type 7 WL nutrient broth was prepared by dissolving 6 g of WL nutrient powder (DIFCO Laboratories, Inc. Detroit, MI) in 100 ml of distilled water. The pH of this media is about 5. 4. To simulate the wine pH, 10% L-malic acid was used to adjust the pH to 3. 4. The broth of both pH values was autoclaved for 20 minutes at 121 °C. WLC media was prepared by adding 1 ml of 0. 5% (w/v) cycloheximide stock solution into 100 ml of sterile WL broth.

Brettanomyces strains 1,2 and 3 isolated from contaminated wine were inoculated into 5 ml of sterile WLC media separately. The cultures were allowed to grow for 1 week at room temperature (about 22 °C) on an Orbit rotary bench top shaker at 180 rpm. All the wells of a microtiter plate were filled with 100 jul of sterile WLC media Stock solutions of 2000 ppm of avidin and Lysozyme type 7 (Canadian Inovatech, Inc. ) were prepared by weighing 0. 1 g of dried powder into 5 ml of sterile WLC media. Then 100 jul of the antimicrobial stock solution was added into each well in the first column of the microtiter plate. Serial doubling dilutions were made of the first column across the microtiter plate. The last column served as a control (no antimicrobial solution).

The cells of the Brettanomyces inoculums were counted on a hemacytometer under a microscope and the inoculum was diluted to 10t cells/ml with WLC media. Then 10 1 of the diluted inoculum was added to each well in the designated rows. The plates were then incubated at room temperature (22°C) for two weeks. Yeast growth on day 7 and 14 was observed.

The minimal inhibitory concentrations (MIC) for the three Brettanomyces strains tested are presented in Figures 1 and 2. Avidin can inhibit the growth of strains 1 and 2 for 2 weeks at a concentration less than 250 ppm at both pH 5. 4 and 3. 4. For Lysozyme type 7, the MIC is less than 100 ppm when tested at pH 5. 4 and 250 ppm when tested at pH 3. 4.

Example 3 Inhibition of Kloeckera bv Ivsozvme avidin and Lysozyme type 7 The preparation of WLC media is the same as in Example 2. Kloeckera, isolated from contaminated wine, was inoculated into 5 ml of sterile WLC media.

The culture was allowed to grow for 2-3 days at room temperature (about 22 °C) on an Orbit rotary bench top shaker at 180 rpm.

All of the wells of a microtiter plate were filled with 100 y1 of sterile WLC media. Stock solutions of 2000 ppm of lysozyme, pure avidin and Lysozyme type 7 (Canadian Inovatech, Inc. ) were prepared in sterile WLC media. Then 100 1 of the antimicrobial stock solution was added into each well of column 1 on the microtiter plate. Serial doubling dilution was made from the first column. The last column was left as a control.

The cells of Kloeckera inoculum were counted on a hemacytometer under the microscope and the inoculum was diluted to 10* cells/ml with WLC media.

Then 10 au of the diluted inoculum was added to each well in the designated rows.

The plates were then incubated at room temperature (22°C) for two weeks. Yeast growth on days 1-3 was observed.

The minimum inhibitory concentration of lysozyme for the Kloeckera strain tested are presented in Figure 3. At pH 5. 4, 500 ppm of lysozyme can inhibit the growth of Kloeckera for 1 day. More than 1000 ppm of lysozyme is required for a longer inhibition period. At pH 3. 4, lysozyme is more effective against Kloeckera. At concentrations of 125,250 and 500 ppm, Kloeckera can be inhibited for 1,2 and 3 days, respectively.

The minimum inhibitory concentrations for Lysozyme type 7 on Kloeckera are presented in Figure 4. Lysozyme type 7 has a higher efficacy in inhibiting Kloeckera than lysozyme alone. At pH 5. 4, 500 ppm of the Lysozyme type 7 can inhibit the yeast growth for 3 days. At pH 3. 4, only 250 ppm of Lysozyme type 7 is required for the same length of inhibition.

Pure avidin also slowed down Kloeckera growth at different concentrations both at pH 5. 4 and 3. 4.

Example 4 Inhibition of Kloeckera bv Ivsozvme and Lysozyme type 7 The preparation of WLC media was the same as in Example 2. Two 10 ml volumes of WLC media were inoculated with Kloeckera (wine isolate). The cultures were allowed to grow for 4 days at room temperature (about 22 °C) on an Orbit rotary bench top shaker (Lab Line, Instruments Inc. , Melrose Park, IL) at 180 rpm. The cells were counted on a hemacytometer under the microscope. The culture was then diluted to 106 cells/ml.

Stock solutions of 2000 ppm of lysozyme and Lysozyme type 7 in WLC media were prepared by weighing 20 mg of the antimicrobials into 10 ml of sterile WLC media.

Table 2 shows the concentration of antimicrobial added to the media containing Kloeckera for testing.

Table 2 Treatment Conc of Vol. of stock Vol. of Vol. of WLC antimicrobialsolution(mi) culture (106 media (ml) (ppm) cells/ml) 1000.1 5 2 1000.250.14.65 3 2500.620.14.28 4 500 1.25 0.1 3.65 5 1000 2.5 0.1 2.4 The yeast cells from each experiment were counted 1,3 and 5 days after inoculation. Dilutions were made whenever it was necessary to get an accurate count. The results for lysozyme are shown in Table 3 and Figure 5.

Table 3 Treatment Lysozyme Time (days) Conc. (ppm) 1 3 5 1 0 2.0x 108 1.6x 108 1.8x 107 2 100 1. 2x10$ 1. 2x108 1. 3x10' 3 250 1. 0 x 10'9. 3 x 107 1. 3 x 107 4 500 6.5x106 7.5x107 7.1x106 1000 6.0 x 105 5.0 x 107 4.1 x 106 The results of treatment with Lysozyme type 7 are shown in Table 4 and Figure 6.

Table 4 Treatment Lysozyme Time (days) type 7 Conc. 1 3 5 (ppm) 1 0 1. 6 x 10'1. 5 x 10'1. 5 x107 2 100 1.5 x 107 1.0 x 108 1.1 x 107 3 250 1. 3x 107 5. 5 x 10'7. 5 x106 4 500 4.0 x 105 3.2 x 107 7.1 x 106 5 1000 2.0 x 105 2.3 x 106 2.9 x 106 Example 5 Inhibition of Brettanomyces by Lysozyme type 7 The preparation of WLC media was the same as described in Example 4.

Two 10 ml WLC media were inoculated with Brettanomyces. The cultures were allowed to grow for 7 days at room temperature (about 22°C) on the Orbit rotary bench top shaker (Lab Line) at 180 rpm. The cells were counted on a

hemacytometer under the microscope. The culture was then diluted to 106 cells/ml.

Stock solutions of 2000 ppm of Lysozyme type 7 in AC media were prepared by weighing 20 mg of Lysozyme type 7 into 10 ml of sterile AC media.

Table 5 shows the concentration of Lysozyme type 7 added to the media containing Brettanomyces for testing.

Table 5 Experiment Conc. of Vol. of stock Vol. of Vol. of WLC antimicrobial solution (ml) culture (106 media (ml) cells/ml) 1 0 0 0.1 5 2 100 0. 25 0. 14. 65 3 250 0.62 0.1 4.28 4 500 1.25 0.1 3.65 5 1000 2. 5 0. 12. 4 The yeast cells from each experiment were counted 1, 3,5 and 7 days after inoculation. Dilutions were made whenever it was necessary to get an accurate count. The results for Lysozyme type 7 are shown Table 6 and Figure 7.

Table 6 Inhibition of Brettanomvces by Lysozvme type 7 three days after inoculation Experiment Lysozyme type 7 Cell growth (cells/ml) concentration (ppm) 1 0 2. 8 x 10' 2100 1. 8x10' 3 250 1.6 x 107 4 500 1.7 x 107 5 1000 1.1 x 107